Project Summary Efforts in recent years have led to a number of breakthroughs in our understanding of how the immune system recognizes HIV-1, and this has brought optimism that we are at, or at least close to, one threshold of success ? the elicitation of broadly neutralizing antibodies (bNAbs) in response to vaccination. Substantial focus of late has shifted toward the design of (SOSIP) immunogens that aim at mimicking the trimeric prefusion closed conformation of Env, since this is a conformation recognized preferentially by bNAbs, but not by weakly and non-neutralizing antibodies. These efforts have resulted in primarily autologous neutralization against the specific immunogen strain. However, when multiple strains are used in the immunization strategy, either as a cocktail or sequentially, some (generally sporadic) heterologous Tier-2 neutralization has been observed. These results serve as a strong indicator that incorporating strain sequence diversity within the immunization strategy will be a critical step toward the design of a vaccine that can elicit truly broadly neutralizing antibody responses. In addition, recent epitope-focused efforts that rely on a prime-boost strategy involving a fusion epitope-peptide as a prime and SOSIP trimer as a boost have led to arguably the strongest elicitation of heterologous bNAb responses to date. While the elicited neutralization breadth was virtually unprecedented, it was still primarily limited to strains that matched the sequence of the immunogen construct that was used in the immunization. Together, these results strongly motivate exploring the premise that the incorporation of strain sequence diversity will be critical for the elicitation of truly broadly neutralizing antibody responses. There are a number of different ways in which strain sequence diversity can be incorporated into the immunization strategy, with immunogen cocktails and sequential immunization being two standard examples. Here, we propose to develop a new technology that will allow the incorporation of multiple diverse antigens onto the surface of a self-assembling nanoparticle. The hypothesis here is that placing antigens from diverse strains in spatial proximity onto the same particle will engage B cells that can simultaneously recognize the sequence-diverse, spatially proximal, spikes. In this application, we propose to develop such multivalent nanoparticles in two contexts: for displaying trimeric Env (Specific Aim 1) and for displaying epitope-peptides (Specific Aim 2) from diverse HIV-1 strains. For both aims, nanoparticles will be designed using computational technology and validated experimentally using a variety of techniques. Top-ranked nanoparticles will be used for immunization in rabbits, to evaluate their ability to elicit robust heterologous bNAb responses. If successful, this proposal will not only generate new lead HIV-1 immunogen candidates, but will also result in the development of novel multivalent nanoparticle platforms that will be of general utility for vaccine design not only for HIV-1, but also for other pathogens of biomedical interest.